Methods and compositions for treating diseases and conditions associated with gonadotropin releasing hormone receptor

ABSTRACT

The disclosure is directed in non-limiting embodiments to compounds, compositions, and methods of treating conditions and diseases associated with activation of the gonadotropin GnRH receptor (GnRHR), particularly those involving GnRHR activating autoantibodies (GnRHR AAbs). In one non-limiting embodiment, the disease is Polycystic Ovary Syndrome (PCOS). The therapeutic compounds in at least certain embodiments include peptides which at least partially comprise D-amino acids, such as retro-inverso D-amino acid (RID) peptides, which are able to bind with high affinity to GnRHR AAbs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. 119(e) of U.S.Provisional Application Ser. No. 62/634,324, filed Feb. 23, 2018, whichis hereby expressly incorporated herein by reference in its entirety.

BACKGROUND

Polycystic Ovary Syndrome (PCOS) is a systemic disease characterized bymultiple ovarian cysts, infertility, pain, discomfort, and associatedmetabolic abnormalities including insulin resistant diabetes mellitus,abnormal sex hormone function, hirsutism, and acne. It is considered ofunknown etiology and frequently occurs in adolescence. PCOS involves thepituitary-ovarian reproductive organs, resulting in anovulatoryinfertility in 8-10% of women of reproductive age. Its causation iscurrently unknown but is associated with abnormal pituitary controland/or release of both luteinizing hormone (LH) and follicle stimulatinghormone (FSH), both of which are intimately related to development andrelease of ova from the ovary in timely fashion for possiblefertilization when a viable sperm is in proximity. In this condition,the release of the developing ova is severely impaired and generally isinternally reabsorbed without release. The developing follicle continuesto be stimulated by this abnormal absence of rupture and release andbecomes a persistent and enlarged cyst leading to distortion of theovarian architecture and further disturbing normal development ofsubsequent follicles.

This infertility is profound in many conditions leading to heartbreak,frustration, self-recrimination, and marital discord. Currentinfertility therapies are frustratingly of low success, and currentstate of the art resides in in vitro fertilization, often requiring aseparate donor and emplacement of the embryo. The costs are very high(e.g., $75K for three attempts) and are without guarantee of issue.Moreover, PCOS is not just an issue of infertility, as a significantmajority of these women develop a complex syndrome over several yearsthat includes obesity, significant insulin resistance manifested by Type2 diabetes mellitus, hypertension, and signs of hyper-androgenism(elevation of testosterone and weak androgens) including acne andhirsutism. A smaller subgroup includes women of thin nature despiteeating freely yet who do not develop the above-mentioned metabolicsyndrome. These women nevertheless share in the infertility andpolycystic ovary presentation. This metabolic syndrome that is soprevalent must be treated using multiple dietary and pharmacologicalapproaches. The diabetes has most commonly been treated with metforminwith modest improvement, despite the frequent side effects of themedication and its long term inability to cure the disease.Interestingly, in some subjects there is minimal or very modestimprovement in the infertility but this is not common. Newer diabetesmedications are being tried but have significant costs and are notrecognized as more effective than metformin on the infertility. Thehypertension is treated with usually more than one antihypertensiveagent with their concomitant cost and side effects. The hyper-androgenstate is of particular concern for women of all ages but is especiallydevastating for young women in society. The therapy usually is startedtoo late and or inadequately and consists of birth control pill (BCP)therapy to suppress LH and the use of androgen receptor antagonists withtheir concurrent side effects and expense. Alterations in gonadotropinreleasing hormone (GnRH) or its receptor (GnRHR) have not beensuccessfully associated with causation.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A shows the ELISA O.D. (vertical axis) directed against theGnRHR-extracellular loop 2 (ECL2) sequence for the PCOS for serum frominfertile but ovulatory control subjects. The values in the left panelwere assayed initially and subsequently repeated after a 5 mo. intervalwith storage at 4° C. The PCOS values were significantly higher than thecontrols on both assays, with a p=0.003 and 0.005, respectively. Theslight decrease in n for the assay in the second panel was due toinsufficient sera in one PCOS subject and two control subjects. Thedashed line represents+2 SD of the controls. The solid line is the meanfor each group.

FIG. 1B shows an ELISA assay of these subjects using the GnRHR-ECL1sequence. There was no evidence for an Ab directed toward this adjacentECL (extracellular loop).

FIG. 1C shows a ROC curve comparing the ELISA OD assayed in the PCOS andcontrol subjects (data are from the right panel of FIG. 1A). There is asensitivity of 72% and a specificity of 55% compared to a theoreticalcontrol population (dashed line). The area under the curve wassignificantly different from the controls (p=0.0013). Diagonal segmentsare produced by ties. These were subjects matched for age and BMR andblinded to the lab.

FIG. 2A shows GnRHR-ECL2 ELISA values for a group of 32 PCOS subjectscompared to the subtype of infertility in the control subjects. Theseassays were run on 96 well plates. The control values were significantlylower than the PCOS subjects in each group.

FIG. 2B shows GnRHR-ECL2 ELISA values for a group of 27 PCOS subjectscompared to the subtype of infertility in the control subjects. Theseassays were run on 96 well plates. The control values were significantlylower than the PCOS subjects in each group.

FIG. 3A shows the dosage response curve for the normal ligand hLHRH(human Leutenizing Hormone Releasing Hormone) on relative Ca²⁺ inducedFluo-8 NW fluorescence. The values are expressed as the percentage ofmaximal fluorescence induced during the exposure period.

FIG. 3B shows the dosage response curve for the synthetic agonistleuprolide on relative Ca²⁺ induced Fluo-8 NW fluorescence. The valuesare expressed as the percentage of maximal fluorescence induced duringthe exposure period. The normal ligand hLHRH (FIG. 3A) and leuprolidedemonstrated similar actions at 10⁻⁹ M and 10⁻⁸ M, n=4, respectively.

FIG. 4A shows a dosage response curve for IgG derived from 3 subjectswith a positive GnRHR-ECL2 ELISA. The significance for each dosage abovebaseline is shown.

FIG. 4B shows the PCOS response of FIG. 4A compared to IgG from 3control subjects.

FIG. 5 demonstrates the effect of the GnRHR specific antagonistcetrorelix (10⁻⁵ M) on the GnRHR activity of 4 subjects with PCOS andtheir controls. The antagonist suppressed the relative fluorescenceexpressed as % of maximum fluorescence to baseline while the activityfor the controls was not significantly changed.

FIG. 6 shows the effect of the GnRHR synthetic agonist leuprolide on theGnRHR transfected cells alone and in the presence of IgG purified fromPCOS subjects with active autoantibodies (AAb). The presence of the IgGincreased the total activity at least in an additive fashion over thebuffer+leuprolide. (n=4, p<0.01).

FIG. 7 shows a minipin ELISA using PCOS serum and its interaction withmultiple pins containing sequences, each offset by two amino acids, ofthe GnRHR-ECL2 peptide structure. The peaks represent sites of increasedbinding by Abs present in the sera. The amino acid sequence for each pinis shown at the bottom. Representative PCOS with 2 dominant peaks. Eachcolor bar represents the time of O.D. reading after addition ofchromagen (30, 60, and 90 min).

FIG. 8 shows a minipin ELISA using PCOS with a single dominant peak andhigh O.D. (note different vertical scale for high O.D.).

FIG. 9 shows a minipin ELISA of a control with single first peak withmoderate O.D. value (note different vertical scale).

FIG. 10 shows a minipin ELISA of a control with relatively low activity(note low vertical scale compared to FIG. 9).

DETAILED DESCRIPTION

The present disclosure is directed, in at least certain embodiments, tocompositions and methods of treating conditions and diseases associatedwith activation of the gonadotropin GnRH receptor (GnRHR), particularly(but not limited to) those involving GnRHR activating autoantibodies(GnRHR AAbs). A particular (but non-limiting) novel feature of thepresent disclosure is the discovery of the relationship of GnRHR AAbsand Polycystic Ovary Syndrome. Therefore, in at least one embodiment,the present disclosure is directed to treatment of PCOS by thetherapeutic clearing of GnRHR AAbs from the circulation. The therapeuticcompounds in at least certain embodiments include peptides at leastpartially comprising D-amino acids, and in further embodiments includeretro-inverso D-amino acid (RID) peptides, which are able to bind withhigh affinity to GnRHR AAbs, thereby inhibiting the binding of the GnRHRAAbs to GnRHR via competitive inhibition.

Without wishing to be bound by theory, it is believed that activatingautoantibodies (GnRHR AAbs) which bind to the extracellular loop 2(ECL2) of GnRHR are produced from an extraneous stimulus (e.g., aninfection, stress, etc.) and interact with hypothalamic/pituitary GnRHreceptors to inappropriately alter the synthesis and periodic release ofLH, leading to hyperandrogenemia, induction of insulin resistance (IR),and metabolic complications.

Before further describing various embodiments of the present disclosurein more detail by way of exemplary description, examples, and results,it is to be understood that the compounds, compositions, and methods ofpresent disclosure are not limited in application to the details ofspecific embodiments and examples as set forth in the followingdescription. The description provided herein is intended for purposes ofillustration only and is not intended to be construed in a limitingsense. As such, the language used herein is intended to be given thebroadest possible scope and meaning; and the embodiments and examplesare meant to be exemplary, not exhaustive. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting unless otherwiseindicated as so. Moreover, in the following detailed description,numerous specific details are set forth in order to provide a morethorough understanding of the present disclosure. However, it will beapparent to a person having ordinary skill in the art that the presentdisclosure may be practiced without these specific details. In otherinstances, features which are well known to persons of ordinary skill inthe art have not been described in detail to avoid unnecessarycomplication of the description. It is intended that all alternatives,substitutions, modifications, and equivalents apparent to those havingordinary skill in the art are included within the scope of the presentdisclosure. Thus, while the compounds, compositions, and methods of thepresent disclosure have been described in terms of particular (butnon-limiting) embodiments, it will be apparent to those of skill in theart that variations may be applied to the compounds, compositions, andmethods, and in the steps or in the sequence of steps of the methodsdescribed herein without departing from the concept, spirit, and scopeof the inventive concepts.

All patents, published patent applications, and non-patent publicationsincluding published articles mentioned in the specification orreferenced in any portion of this application, are herein expresslyincorporated by reference in their entirety to the same extent as ifeach individual patent or publication was specifically and individuallyindicated to be incorporated by reference.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those having ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities, and plural terms shall include the singular. Where usedherein, the specific term “single” is limited to only “one.”

As utilized in accordance with the methods, compounds, and compositionsof the present disclosure, the following terms, unless otherwiseindicated, shall be understood to have the following meanings:

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The use of the term “or” in the claims isused to mean “and/or” unless explicitly indicated to refer toalternatives only or when the alternatives are mutually exclusive,although the disclosure supports a definition that refers to onlyalternatives and “and/or.” The use of the term “at least one” will beunderstood to include one as well as any quantity more than one,including but not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30,40, 50, 100, or any integer inclusive therein. The term “at least one”may extend up to 100 or 1000 or more, depending on the term to which itis attached; in addition, the quantities of 100/1000 are not to beconsidered limiting, as higher limits may also produce satisfactoryresults. In addition, the use of the term “at least one of X, Y, and Z”will be understood to include X alone, Y alone, and Z alone, as well asany combination of X, Y, and Z.

As used herein, all numerical values or ranges include fractions of thevalues and integers within such ranges and fractions of the integerswithin such ranges unless the context clearly indicates otherwise. Thus,to illustrate, reference to a numerical range, such as 1-10 includes 1,2, 3, 4, 5, 6, 7, 8, 9, 10, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc.,and so forth. Reference to a range of 1-50 therefore includes 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc., upto and including 50, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., 2.1, 2.2,2.3, 2.4, 2.5, etc., and so forth. Reference to a series of rangesincludes ranges which combine the values of the boundaries of differentranges within the series. Thus, to illustrate reference to a series ofranges, for example, of 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75,75-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750,750-1,000, includes ranges of 1-20, 10-50, 50-100, 100-500, and500-1,000, for example. Reference to an integer with more (greater) orless than includes any number greater or less than the reference number,respectively. Thus, for example, reference to less than 100 includes 99,98, 97, etc. all the way down to the number one (1); and less than 10includes 9, 8, 7, etc. all the way down to the number one (1).

As used in this specification and claims, the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C, or combinations thereof” is intended to includeat least one of: A, B, C, AB, AC, BC, or ABC, and if order is importantin a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AAB, BBC,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the composition, themethod used to administer the composition, or the variation that existsamong the study subjects. As used herein the qualifiers “about” or“approximately” are intended to include not only the exact value,amount, degree, orientation, or other qualified characteristic or value,but are intended to include some slight variations due to measuringerror, manufacturing tolerances, stress exerted on various parts orcomponents, observer error, wear and tear, and combinations thereof, forexample. The term “about” or “approximately,” where used herein whenreferring to a measurable value such as an amount, a temporal duration,and the like, is meant to encompass, for example, variations of ±20% or±10%, or ±5%, or ±1%, or ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods and asunderstood by persons having ordinary skill in the art. As used herein,the term “substantially” means that the subsequently described event orcircumstance completely occurs or that the subsequently described eventor circumstance occurs to a great extent or degree. For example, theterm “substantially” means that the subsequently described event orcircumstance occurs at least 90% of the time, or at least 95% of thetime, or at least 98% of the time.

As used herein any reference to “one embodiment” or “an embodiment”means that a particular element, feature, structure, or characteristicdescribed in connection with the embodiment is included in at least oneembodiment and may be included in other embodiments. The appearances ofthe phrase “in one embodiment” in various places in the specificationare not necessarily all referring to the same embodiment and are notnecessarily limited to a single or particular embodiment. Further, allreferences to one or more embodiments or examples are for purposes ofillustration only and are to be construed as non-limiting of the claims.

The term “pharmaceutically acceptable” refers to compounds andcompositions which are suitable for administration to humans and/oranimals without undue adverse side effects such as (but not limited to)toxicity, irritation, and/or allergic response commensurate with areasonable benefit/risk ratio. The compounds or conjugates of thepresent disclosure may be combined with one or morepharmaceutically-acceptable excipients, including carriers, vehicles,and diluents which may improve solubility, deliverability, dispersion,stability, and/or conformational integrity of the compounds orconjugates thereof.

By “biologically active” is meant the ability to modify thephysiological system of an organism without reference to how the activeagent has its physiological effects.

As used herein, “pure” or “substantially pure” means an object speciesis the predominant species present (i.e., on a molar basis it is moreabundant than any other object species in the composition thereof), andparticularly a substantially purified fraction is a composition whereinthe object species comprises at least about 50 percent (on a molarbasis) of all macromolecular species present. Generally, a substantiallypure composition will comprise more than about 80% of all macromolecularspecies present in the composition, more particularly more than about85%, more than about 90%, more than about 95%, or more than about 99%.The term “pure” or “substantially pure” also refers to preparationswhere the object species is at least 60% (w/w) pure, or at least 70%(w/w) pure, or at least 75% (w/w) pure, or at least 80% (w/w) pure, orat least 85% (w/w) pure, or at least 90% (w/w) pure, or at least 92%(w/w) pure, or at least 95% (w/w) pure, or at least 96% (w/w) pure, orat least 97% (w/w) pure, or at least 98% (w/w) pure, or at least 99%(w/w) pure, or 100% (w/w) pure.

Non-limiting examples of animals or subjects within the scope andmeaning of this term include dogs, cats, rats, mice, guinea pigs,chinchillas, horses, goats, cattle, sheep, zoo animals, Old and NewWorld monkeys, non-human primates, and humans.

“Treatment” refers to therapeutic treatments. “Prevention” refers toprophylactic or preventative treatment measures or reducing the onset ofa condition or disease. The term “treating” refers to administering thecomposition to a subject for therapeutic purposes and/or for prevention.

The terms “therapeutic composition” and “pharmaceutical composition”refer to an active agent-containing composition that may be administeredto a subject by any method known in the art or otherwise contemplatedherein, wherein administration of the composition brings about atherapeutic effect as described elsewhere herein. In addition, thecompositions of the present disclosure may be designed to providedelayed, controlled, extended, and/or sustained release usingformulation techniques which are well known in the art.

The term “effective amount” refers to an amount of an active agent whichis sufficient to exhibit a detectable therapeutic or treatment effect ina subject without excessive adverse side effects (such as (but notlimited to) substantial toxicity, irritation, and allergic response)commensurate with a reasonable benefit/risk ratio when used in themanner of the present disclosure. The effective amount for a subjectwill depend upon the subject's type, size and health, the nature andseverity of the condition to be treated, the method of administration,the duration of treatment, the nature of concurrent therapy (if any),the specific formulations employed, and the like. Thus, it is notpossible to specify an exact effective amount in advance. However, theeffective amount for a given situation can be determined by one ofordinary skill in the art using routine experimentation based on theinformation provided herein.

The term “ameliorate” means a detectable or measurable improvement in asubject's condition, disease or symptom thereof. A detectable ormeasurable improvement includes a subjective or objective decrease,reduction, inhibition, suppression, limit or control in the occurrence,frequency, severity, progression, or duration of the condition ordisease, or an improvement in a symptom or an underlying cause or aconsequence of the disease, or a reversal of the disease. A successfultreatment outcome can lead to a “therapeutic effect,” or “benefit” ofameliorating, decreasing, reducing, inhibiting, suppressing, limiting,controlling or preventing the occurrence, frequency, severity,progression, or duration of a disease or condition, or consequences ofthe disease or condition in a subject.

A decrease or reduction in worsening, such as stabilizing the conditionor disease, is also a successful treatment outcome. A therapeuticbenefit therefore need not be complete ablation or reversal of thedisease or condition, or any one, most or all adverse symptoms,complications, consequences or underlying causes associated with thedisease or condition. Thus, a satisfactory endpoint may be achieved whenthere is an incremental improvement such as a partial decrease,reduction, inhibition, suppression, limit, control or prevention in theoccurrence, frequency, severity, progression, or duration, or inhibitionor reversal of the condition or disease (e.g., stabilizing), over ashort or long duration of time (hours, days, weeks, months, etc.).Effectiveness of a method or use, such as a treatment that provides apotential therapeutic benefit or improvement of a condition or disease,can be ascertained by various methods and testing assays.

The following abbreviations may be used herein for amino acids:alanine:ala:A; arginine:arg:R; asparagine:asn:N; aspartic acid:asp:D;cysteine:cys:C; glutamic acid:glu:E; glutamine:gln:Q; glycine:gly:G;histidine:his:H; isoleucine:ile:I; leucine:leu:L; lysine:lys:K;methionine:met:M; phenylalanine:phe:F; proline:pro:P; serine:ser:S;threonine:thr:T; tryptophan:trp:W; tyrosine:tyr:Y; and valine:val:V.

The term “peptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids to forman amino acid sequence. In certain embodiments, the peptides can rangein length from 4 to 10 to 15 to 25 to 40 to 60 to 75 to 100 amino acids,for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids.

The term “polypeptide” or “protein” is used herein to designate a seriesof amino acid residues, connected one to the other typically by peptidebonds between the alpha-amino and carbonyl groups of the adjacent aminoacids, wherein the length is longer than a single peptide. A peptideconjugate refers, in a non-limiting embodiment, to a compound comprisinga peptide of the present disclosure which is conjugated (e.g.,covalently linked, directly or indirectly via a linker sequence) toanother molecule, such as (but not limited to) a carrier molecule suchas (but not limited to) a protein or other polymeric molecule, e.g., aserum albumin molecule or antibody, or other therapeutic compound suchas (but not limited to) a drug, or an imaging or diagnostic moiety, andwherein the peptide retains its activity (e.g., binding, targeting,imaging, or inhibitory) even when conjugated to the molecule. Thepeptides of the present disclosure may be produced using any nucleotidesequence which encodes the desired amino acid sequence. Any of thepeptides described herein or active variants thereof may be used to makethe peptide conjugates of the present disclosure.

Peptides of the present disclosure and the nucleic acids which encodethem include peptide and nucleic acid variants which comprisesubstitutions (conservative or non-conservative) of the native aminoacids or bases. For example, the peptide variants include, but are notlimited to, variants that are not exactly the same as the sequencesdisclosed herein, but which have, in addition to the substitutionsexplicitly described for various sequences listed herein, additionalsubstitutions of amino acid residues (conservative or non-conservative)which substantially do not impair the activity or properties of thevariants described herein. Examples of such conservative amino acidsubstitutions may include, but are not limited to, ala to gly, ser, orthr; arg to gln, his, or lys; asn to asp, gln, his, lys, ser, or thr;asp to asn or glu; cys to ser; gln to arg, asn, glu, his, lys, or met;glu to asp, gln, or lys; gly to pro or ala; his to arg, asn, gln, ortyr; ile to leu, met, or val; leu to ile, met, phe, or val; lys to arg,asn, gln, or glu; met to gln, ile, leu, or val; phe to leu, met, trp, ortyr; ser to ala, asn, met, or thr; thr to ala, asn, ser, or met; trp tophe or tyr; tyr to his, phe or trp; and val to ile, leu, or met.

One of ordinary skill in the art would readily know how to make,identify, select, or test such variants for receptor targeting activityagainst the same receptors targeted by the native peptides. Particularexamples of conservative amino acid substitutions include, but are notlimited to, gly:ala substitutions; val:ile:leu substitutions;asn:glu:his substitutions; asp:glu substitutions; ser:thr:metsubstitutions; lys:arg:his substitutions; and phe:tyr:trp substitutions.Other types of substitutions, variations, additions, deletions, andderivatives that result in functional variant peptides are alsoencompassed by the present disclosure, and one of skill in the art wouldreadily know how to make, identify, or select such variants orderivatives, and how to test for receptor binding activity of thosevariants.

The term “homologous” or “% identity” as used herein means a nucleicacid (or fragment thereof) or a peptide having a degree of homology tothe corresponding natural reference nucleic acid or peptide that may bein excess of 60%, or in excess of 65%, or in excess of 70%, or in excessof 75%, or in excess of 80%, or in excess of 85%, or in excess of 90%,or in excess of 91%, or in excess of 92%, or in excess of 93%, or inexcess of 94%, or in excess of 95%, or in excess of 96%, or in excess of97%, or in excess of 98%, or in excess of 99%, or other specificpercentages described herein. For example, in regard to peptides, thepercentage of homology or identity as described herein is typicallycalculated as the percentage of amino acid residues found in the smallerof the two sequences which align with identical amino acid residues inthe sequence being compared, when four gaps per 100 amino acids may beintroduced to assist in that alignment (as set forth by Dayhoff, inAtlas of Protein Sequence and Structure, Vol. 5, p. 124, NationalBiochemical Research Foundation, Washington, D.C. (1972)). In oneembodiment, the percentage homology as described above is calculated asthe percentage of the components found in the smaller of the twosequences that may also be found in the larger of the two sequences(with the introduction of gaps), with a component being defined as asequence of four, contiguous amino acids. Also included as substantiallyhomologous is any protein product which may be isolated by virtue ofcross-reactivity with antibodies to the native protein product. Sequenceidentity or homology can be determined by comparing the sequences whenaligned so as to maximize overlap and identity while minimizing sequencegaps. In particular, sequence identity may be determined using any of anumber of mathematical algorithms. A non-limiting example of amathematical algorithm used for comparison of two sequences is thealgorithm of Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1990, 87,2264-2268, modified as in Karlin & Altschul, Proc. Natl. Acad. Sci. USA1993, 90, 5873-5877.

In at least one embodiment, “% identity” represents the number of aminoacids or nucleotides which are identical at corresponding positions intwo sequences of a peptide or nucleic acids encoding similar peptides.For example, two amino acid sequences each having 15 residues will haveat least 60% identity when at least 9 of the amino acids atcorresponding positions are the same, at least 66% identity when atleast 10 of the amino acids at corresponding positions are the same, atleast 73% identity when at least 11 of the amino acids at correspondingpositions are the same, at least 80% identity when at least 12 of theamino acids at corresponding positions are the same, at least 86%identity when at least 13 of the amino acids at corresponding positionsare the same, and at least 93% identity when at least 14 of the aminoacids at corresponding positions are the same. In another example, twoamino acid sequences each having 19 residues will have at least 73%identity when at least 14 of the amino acids at corresponding positionsare the same, at least 78% identity when at least 15 of the amino acidsat corresponding positions are the same, at least 84% identity when atleast 16 of the amino acids at corresponding positions are the same, atleast 89% identity when at least 17 of the amino acids at correspondingpositions are the same, and at least 94% identity when at least 18 ofthe amino acids at corresponding positions are the same.

Similarly, two amino acid sequences each having 20 residues will have atleast 95% identity when 19 of the amino acids at corresponding positionsare the same, or at least 90% identity when at least 18 of the aminoacids at corresponding positions are the same, or at least 85% identitywhen at least 17 of the amino acids at corresponding positions are thesame, or at least 80% identity when at least 16 of the amino acids atcorresponding positions are the same. In other non-limiting examples,two amino acid sequences each having 100 residues will have 95% identitywhen 95 of the amino acids at corresponding positions are the same. Twoamino acid sequences each having 100 residues will have at least 90%identity when at least 90 of the amino acids at corresponding positionsare the same. Further, where a sequence is described herein as having“at least X % identity to” a reference sequence, this is intended toinclude, unless indicated otherwise, all percentages greater than X %,such as for example, (X+1)%, (X+2)%, (X+3)%, (X+4)%, and so on, up to100%.

Demonstrated for the first time herein is a viable pathophysiology thatexplains the causation of PCOS and its relationship to onset at a youngage in females that are most susceptible to autoimmune issues. It isfurther believed that this condition afflicts males whose male organsare less susceptible to cyst development but whose androgen andresulting acne and hirsutism may be subject to these influences.Further, a non-genetic form of hypogonadotropic hypogonadism is presentin both young and elderly males, caused by such autoantibodies. Therelationship of GnRHR-ECL-AAB to the Metabolic Syndrome in 80% of femalesubjects with PCOS raises the important likelihood that this samerelationship exists in some percentage of males who have acquiredMetabolic Syndrome. This is a predictable occurrence given that theautoantibodies such as AAb are not a respecter of sexual status otherthan occurring at a higher percentage of females than males. In summary,this reflects an entirely new direction and course of treatment for PCOSand related issues in hyper-androgenic states that did not exist priorto the therapeutic embodiments of the present disclosure.

In at least certain embodiments, the present disclosure is directed to apeptide comprising 4 to 45 amino acids, wherein the 4 to 45 amino acidscomprise a retro-inverso amino acid sequence of SEQ ID NO:15. Thepeptide may be disposed in a pharmaceutically-acceptable carrier,vehicle, and/or diluent.

In at least certain embodiments, the present disclosure is directed to apeptide comprising 6 to 45 amino acids, wherein the 6 to 45 amino acidscomprise an amino acid sequence of SEQ ID NO:14, at least one D-aminoacid upstream of the N-terminal end of SEQ ID NO:14, and at least oneD-amino acid downstream of the C-terminal end of SEQ ID NO:14. In aparticular (but non-limiting) embodiment, the at least one upstreamD-amino acid is immediately upstream of the N-terminal end of SEQ IDNO:14, and the at least one downstream D-amino acid is immediatelydownstream of the C-terminal end of SEQ ID NO:14. The peptide may bedisposed in a pharmaceutically-acceptable carrier, vehicle, and/ordiluent.

In at least certain embodiments, the present disclosure is directed to amethod of treating a disease or condition associated with thegonadotropin releasing hormone receptor, comprising the step of:administering to a subject in need of such therapy at least one of anyof the peptides described herein above or otherwise contemplated herein.The peptide may be administered in a composition comprising apharmaceutically-acceptable carrier, vehicle, and/or diluent. In atleast one embodiment of the method, the disease or condition isPolycystic Ovary Syndrome (PCOS). In at least one embodiment of themethod, the disease is a condition directly related to (consequent to)Polycystic Ovary Syndrome in males; non-limiting examples of suchconditions include (but are not limited to) GnRHR-AAb positive MetabolicSyndrome with abnormal LH, testosterone, insulin resistance, diabetesmellitus, elevated lipids, and elevated cardiovascular risk. In at leastone embodiment of the method, the disease or condition is a refractorycancer selected from the group consisting of ovarian, breast,endometrial, uterine, prostate, and testicular cancer.

In at least certain embodiments, the present disclosure is directed toan assay for detecting a gonadotropin releasing hormone receptoractivating autoantibody (GnRHR AAb), comprising: a test surface having apeptide bound thereto, the peptide comprising an amino acid sequence ofSEQ ID NO:1, or a portion thereof able to bind with high affinity to theGnRHR AAb. The portion of SEQ ID NO:1 may be selected from the groupconsisting of SEQ ID NOS:1-14 and 16-39.

In at least certain embodiments, the present disclosure is directed to amethod of measuring activity of gonadotropin releasing hormone receptoractivating autoantibodies (GnRHR AAbs) in a purified serum sample,comprising the steps of: (1) providing a test surface withGnRHR-transfected cells disposed thereon; (2) providing a serum samplesuspected of containing GnRHR AAbs, and treating the GnRHR-transfectedcells on the test surface with the serum sample in the presence andabsence of a GnRHR blocker; (3) measuring GnHRH activity of the treatedGnRHR-transfected cells; and (4) comparing the GnHRH activity measuredin the GnRHR-transfected cells treated with the serum sample in thepresence of the GnRHR blocker to the GnHRH activity measured in theGnRHR-transfected cells treated with the serum sample in the absence ofthe GnRHR blocker. The GnRHR blocker may be cetrorelix. TheGnRHR-transfected cells may be Chem-1 cells.

In certain embodiments, by measuring the direct stimulating activitybefore and after adding a specific blocker (e.g., cetrorelix), it can beshown that subjects with PCOS have suppressible activity assays whilecontrols do not, thus providing an alternative diagnostic assay to theELISA. In certain embodiments, by measuring the effect of the AAbs onthe stimulating ability of a synthetic GnRHR agonist (e.g., leuprolide),and likely the natural ligand (LHRH) in the activity assay, the GnRHRAAbs enhance the activity of the agonist while control subjects do notshow this outcome. This effect can be used to enhance the specificity ofthe diagnostic assay. Further, by combining the GnRHR-ECL2-AAb ELISAassay results with results of the AMH-ELISA assay described elsewhereherein, specificity of a PCOS diagnostic assay is further enhanced.

In at least one embodiment of the present disclosure, RID peptidehomologs (retro-inverso enantiomers comprising D-amino acids in place ofthe L-amino acids, and in reverse order) based on the core amino acidsequence VTHC (SEQ ID NO:14) can be used to preferentially bindcompetitively to the GnRHR-AAbs forming a GnRHR-AAb-RID peptide complex,causing the AAb-RID complex to be cleared by the R-E cells lining theliver and the spleen to engulf the complex and eliminate it from thecirculation. The RID peptide thus is a decoy peptide that draws down theAAb levels and decreases/eliminates the GnRHR-AAb from reaching andbinding to the GnRHR receptor. For example, the RID version of VTHC isrepresented by CDHDTDVD (SEQ ID NO:15), where the subscript D representsa D-amino acid, i.e., CD, HD, TD, and VD are D-amino acid enantiomers ofthe natural L-amino acids C, H, T, and V.

In at least one embodiment, the RID peptides are 5-10 amino acidresidues, and comprise any retro-inverso D-amino acid homolog based on acontiguous sequence of FSQCVTHCSFSQ (SEQ ID NO:16) and which includes atleast the RID version of the sequence VTHC, i.e., CDHDTDVD. Examples ofL-amino acid sequences derived from FSQCVTHCSFSQ which can be used astemplates to form such a RID peptide include, but are not limited to:CVTHC (SEQ ID NO:17), QCVTHC (SEQ ID NO:18), SQCVTHC (SEQ ID NO:19),FSQCVTHC (SEQ ID NO:20), VTHCS (SEQ ID NO:21), VTHCSF (SEQ ID NO:22),VTHCSFS (SEQ ID NO:23), VTHCSFSQ (SEQ ID NO:24), CVTHCS (SEQ ID NO:25),QCVTHCS (SEQ ID NO:26), SQCVTHCS (SEQ ID NO:27), FSQCVTHCS (SEQ IDNO:28), CVTHCSF (SEQ ID NO:29), QCVTHCSF (SEQ ID NO:30), SQCVTHCSF (SEQID NO:31), FSQCVTHCSF (SEQ ID NO:32), CVTHCSFS (SEQ ID NO:33), QCVTHCSFS(SEQ ID NO:34), SQCVTHCSFS (SEQ ID NO:35), FSQCVTHCSFS (SEQ ID NO:36),CVTHCSFSQ (SEQ ID NO:37), QCVTHCSFSQ (SEQ ID NO:38), and SQCVTHCSFSQ(SEQ ID NO:39).

The D-amino acid-containing peptides of the present disclosure possessmany advantages over the prior art, including but not limited to: (1)the ability to withstand all natural peptidases which can act onpeptides comprising L-amino acids; (2) the capability for withstandinggastro-enteric exposure to natural peptidases and to limited acidhydrolysis; and (3) the potential for oral administration.

In another embodiment, the therapeutic peptides of the presentdisclosure are mixed L-amino acid/D-amino acid peptides which comprisesa binding epitope which is constructed of an L-amino acid epitope (e.g.,any one of SEQ ID NOS:1-14 and 16-39), while upstream and downstreamportions of the peptide comprise one or more flanking D-amino acids oneach end of the L-amino acid portion of the peptide. These flankingD-amino acids serve to protect the natural L-amino acid epitope portionof the peptide. For example, this embodiment of a D-aminoacid-containing peptide may have the structure:D₁-L-D₂wherein “L” represents the specific natural L-amino acid epitope (e.g.,any one of SEQ ID NOS:1-14 or 16-39), D₁ represents one or more upstreamD-amino acids, and D₂ represents one or more downstream D-amino acids.For example, but not by way of limitation, each of D₁ and D₂ may be 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, ormore D-amino acids, including any D-amino acid version of the 20 naturalL-amino acids.

The compositions of the present disclosure may further contain aconjugate of any of the D-amino acid-containing peptides disclosed,described, or otherwise contemplated herein associated with a labelingagent. Various methods of labeling peptides are known in the art and maybe used in accordance with the present disclosure. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionuclides, fluorescent labels, chemiluminescentlabels, and the like. In some embodiments, the labeling agent may beattached to the peptide by a spacer arm of various lengths to reducepotential steric hindrance. In addition, the terms “label,” “labelingagent,” “detectable marker,” “detection moiety,” and “reporter molecule”are used interchangeably herein. These conjugates are useful in variousdiagnostic methods, as discussed in more detail elsewhere herein.

Therapeutic Uses

Peptide compositions of the present disclosure may be administered intherapeutically effective amounts to treat the various conditionsidentified herein which are associated with the GnRHR, such as (but notlimited to) PCOS. An effective amount is a dosage of the compositionsufficient to provide a therapeutically or medically desirable result oreffect in the subject to which the composition is administered. Theeffective amount will vary with the particular condition being treated,the age and physical condition of the subject being treated, theseverity of the condition, the duration of the treatment, the nature ofthe concurrent or combination therapy (if any), the specific route ofadministration, and like factors within the knowledge and expertise ofthe health practitioner. For example, in connection with methodsdirected towards treating subjects having a condition characterized byPCOS, an effective amount would be an amount sufficient to mitigate,reduce, modulate, inhibit, or otherwise effectively treat one or more ofthe conditions in the subject which are associated with PCOS, including(but not limited to) obesity, insulin resistance as manifested by Type 2diabetes mellitus, hypertension, and hyper-androgenism (elevation oftestosterone and weak androgens) including acne and hirsutism.

GnRHRs are known to be present in peripheral tissues of the femalereproductive tract, such as (but not limited to) the ovaries andendometrium. Over 50% of resistant tumors of the breast and prostate arealso reported to harbor GnRHR. Therefore, in at least certainembodiments, the present disclosure is directed to the treatment ofcancers, particularly (but not limited to) refractory cancers,associated with the presence of GnRHR-activating autoantibodies,including (but not limited to) breast, ovarian, endometrial, uterine,testicular, and prostate cancers. This technology permits development ofpatient selective (precise) therapeutic removal of the deleteriousautoantibodies. The AAbs can be cleared from the systemic circulation bythe GnRHR AAb-binding peptides described elsewhere herein.

Generally, a therapeutically effective amount will vary with thesubject's age, condition, and sex, as well as the nature and extent ofthe disease in the subject, all of which can be determined by one ofordinary skill in the art. The dosage may be adjusted by the individualphysician or veterinarian, particularly in the event of anycomplication. A therapeutically effective amount is typically, but notlimited to, an amount in a range from 0.1 μg/kg to about 2000 mg/kg, orfrom 1.0 μg/kg to about 1000 mg/kg, or from about 0.1 mg/kg to about 500mg/kg, or from about 1.0 mg/kg to about 100 mg/kg, in one or more doseadministrations daily, for one or more days. If desired, the effectivedaily dose of the active compound may be administered as two, three,four, five, six, or more sub-doses, for example, administered separatelyat appropriate intervals throughout the day, optionally, in unit dosageforms. In some embodiments, the compositions are administered for morethan 7 days, more than 10 days, more than 14 days, or more than 20 days.In still other embodiments, the peptide is administered over a period ofweeks or months. In still other embodiments, the peptide is delivered onalternate days, for example, the agent may be delivered every two days,or every three days, or every four days, or every five days, or everysix days, or every week, or every month.

The peptide compositions may be administered alone or in combinationwith the above-described drug therapies and may be administered by avariety of administration routes. The particular mode selected willdepend upon the compound selected, the condition being treated, theseverity of the condition, whether the treatment is therapeutic orprophylactic, and the dosage required for efficacy. The methods of thepresent disclosure, generally speaking, may be practiced using any modeof administration that is medically acceptable, meaning any mode thatproduces effective levels of the active compounds without causingclinically unacceptable adverse effects. The administration may, forexample, be oral, intraperitoneal, intra-cavity such as (but not limitedto) rectal or vaginal, transdermal, topical, nasal, inhalation, mucosal,interdermal, or parenteral routes. The term “parenteral” includessubcutaneous, intravenous, intramuscular, or infusion. Intravenous orintramuscular routes may not be particularly suitable for long termtherapy and prophylaxis. In certain embodiments, however, it may beappropriate to administer the compound in a continuous infusion everyseveral days, or once a week, or every several weeks, or once a month.Intravenous or intramuscular routes may be particularly used inemergency situations. Oral administration may be used for prophylactictreatment because of the convenience to the patient as well as thedosing schedule. Likewise, sustained release devices may be useful incertain embodiments for prophylactic or post-surgery treatment, forexample.

Particular pharmaceutical formulations of the presently disclosedpeptide compositions include, but are not limited to, sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Non-limiting examplesof non-aqueous solvents include propylene glycol, polyethylene glycol,vegetable oils such as (but not limited to) olive oil, and injectableorganic esters such as (but not limited to) ethyl oleate. Aqueouscarriers include water, alcoholic/aqueous solutions, emulsions, orsuspensions, including saline and buffered media. Parenteral vehiclesinclude, for example, sodium chloride solution, Ringer's dextrose,dextrose and sodium chloride, lactated Ringer's, or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example (but not by way of limitation), antimicrobials,anti-oxidants, chelating compounds, inert gases, and the like. Thepharmaceutical compositions may conveniently be presented in unit dosageform and may be prepared by any of the methods well known in the art ofpharmacy.

Compositions suitable for oral administration may comprise discreteunits, such as (but not limited to) capsules, tablets, and lozenges,each containing a predetermined amount of the peptide composition. Othercompositions include suspensions in aqueous liquids or non-aqueousliquids such as (but not limited to) syrup, an elixir, or an emulsion.In yet other embodiments, the particular vehicle is a biocompatiblemicroparticle or implant that is suitable for implantation into themammalian recipient.

Other embodiments of the peptide compositions include pharmaceuticallyacceptable compositions which comprise a therapeutically-effectiveamount of one or more of the compounds described herein, formulatedtogether with one or more pharmaceutically acceptable carriers(additives) and/or diluents. As described in detail below, thepharmaceutically acceptable compositions may be specially formulated foradministration in solid or liquid form, including, but not limited to,those adapted for the following: oral administration, for example,aqueous or non-aqueous solutions or suspensions, tablets, e.g., thosetargeted for buccal, sublingual, and systemic absorption, boluses,powders, granules, pastes for application to the tongue; parenteraladministration, for example, by subcutaneous, intramuscular, intravenousor epidural injection as, for example, a sterile solution or suspension,or sustained-release formulation; topical application, for example, as acream, ointment, or a controlled-release patch or spray applied to theskin; intravaginal or intrarectal administration, for example, as acream or foam; sublingual administration; ocular administration;transdermal administration; or nasal administration.

Some examples of materials which can serve aspharmaceutically-acceptable carriers include, but are not limited to:sugars, such as lactose, glucose, and sucrose; starches, such as cornstarch and potato starch; cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose, and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients, such as cocoabutter and suppository waxes; oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil, and soybean oil;glycols, such as propylene glycol; polyols, such as glycerin, sorbitol,mannitol, and polyethylene glycol; esters, such as ethyl oleate andethyl laurate; agar; buffering agents, such as magnesium hydroxide andaluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters,polycarbonates and/or polyanhydrides; and other non-toxic compatiblesubstances employed in pharmaceutical formulations.

In solid dosage forms of the peptide compositions for oraladministration (capsules, tablets, pills, powders, granules, and thelike), the compound or compounds may be mixed with one or morepharmaceutically-acceptable carriers, including, but not limited to,sodium citrate or dicalcium phosphate, and/or any of the following:fillers or extenders, such as starches, lactose, sucrose, glucose,mannitol, and/or silicic acid; binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose, and/or acacia; humectants, such as glycerol; disintegratingagents, such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate; solutionretarding agents, such as paraffin; absorption accelerators, such asquaternary ammonium compounds; wetting agents, such as cetyl alcohol,glycerol monostearate, and non-ionic surfactants; absorbents, such askaolin and bentonite clay; lubricants, such a talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof; and coloring agents. In the case of capsules,tablets, and pills, the pharmaceutical compositions may also comprisebuffering agents. Solid compositions of a similar type may also beemployed as fillers in soft and hard-shelled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like.

Liquid dosage forms for oral administration of the peptides include, butare not limited to, pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups, and elixirs. In additionto the active ingredient, the liquid dosage forms may contain inertdiluents commonly used in the art, such as, for example (but not by wayof limitation), water or other solvents, solubilizing agents, andemulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurylalcohol, polyethylene glycols, and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, the oral compositions can alsoinclude adjuvants such as (but not limited to) wetting agents,emulsifying and suspending agents, sweetening, flavoring, coloring,perfuming, and preservative agents. Suspensions, in addition to theactive compounds, may contain suspending agents as, for example,ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitanesters, microcrystalline cellulose, aluminum metahydroxide, bentonite,agar-agar, and tragacanth, and mixtures thereof.

EXAMPLES

Examples are provided hereinbelow. However, the present disclosure is tobe understood to not be limited in its application to the specificexperimentation, results, and laboratory procedures disclosed hereinafter. Rather, the Examples are simply provided as one of variousembodiments and are meant to be exemplary, not exhaustive.

Example 1: Activating Antibodies Directed to the ECL2 of GonadotropinReleasing Hormone Receptor are Diagnostic of PCOS

As noted above, PCOS is a systemic disease characterized by ovulatorydysfunction, hyperandrogenism, and polycystic-appearing ovaries ontransvaginal ultrasound with prevalence estimates of up to 19% of thefemale population of reproductive age using the Rotterdam Conferencecriteria. In ovulatory women, gonadotropin-releasing hormone (GnRH) isreleased in a pulsatile manner from the hypothalamus resulting inpulsatile secretion of both LH and FSH, which then communicate with theovaries to result in ovulation. In contrast, PCOS is characterized by avariable and erratic elevation of LH of unknown etiology. PCOS is alsoassociated with significant metabolic sequelae that lead to othersignificant long-term healthcare issues noted above.

The present work was based on a hypothesis that GnRHR activatingantibodies directed to the ECL2 of GnRHR type 1, a G-protein coupledreceptor, would be present in PCOS patients, would contribute to itspathophysiology including erratic LH secretion and downstream effects,and possess diagnostic value.

Methodology

This study was approved by the University of Oklahoma Health SciencesCenter Institutional Review Board as conforming to the overlyingprinciples operative in the United States.

Clinical Subjects: Infertile PCOS subjects based on Rotterdam criteriaand infertile ovulatory controls seen at an academic infertility clinic2012-2016 were included in the study if they had stored serum prior tobeginning fertility treatment. The PCOS subjects were diagnosed based on2 of the 3 Rotterdam criteria (ovulatory dysfunction, clinical orlaboratory evidence of hyperandrogenism, and/or polycystic appearingovaries on transvaginal ultrasound) while ruling out other causes ofovulatory dysfunction (i.e. hypothyroidism, hyperprolactinemia). Thecontrol patients were infertile ovulatory patients being seen forinfertility in the same clinic from 2012-2016. 80 PCOS patients werefirst identified, and then controls were chosen as 1:1 match forrace/ethnicity, age±3 years, and BMI±5. However, controls were excludedif there was no stored serum from initial consultation prior tobeginning infertility treatment. Patients underwent a blood draw attheir initial consultation for routine infertility evaluation. Separatedserum was frozen and stored at −80° C. This serum was de-identified andsent frozen to the laboratory.

Preparation of Specimens: Sera stored at −80° C. were thawed the dayprior to assay in a 4° C. refrigerator. When indicated, IgG was purifiedfrom 0.5 ml sera using ion exchange chromatography (Pierce NAb ProteinA/G Spin Kit, Thermo Scientific, Radnor, Pa.). Protein content wasestimated using spectroscopy at 104 nm and normalized to mcg/ml tostandardize dosage effects. This IgG was used to demonstrate that theobserved activity was related to the autoantibodies and not to otherserum components.

Laboratory Assays: ELISA was performed using a 28-mer peptide having theamino acid sequence SEQ ID NO:1 (DSSGQTKVFSQCVTHCSFSQWWHQAFYN)corresponding to the amino acid sequence of the ECL2 of human GnRHR typeI. This peptide was synthesized and purified to 95% at GenScript(Piscataway, N.J.) and used to coat 96-well (and subsequently 384-well)ELISA plates overnight at a concentration of 10 μg/mL in a coatingbuffer at 4° C. Sera were diluted 1:50 with buffer and added to thewells with incubation overnight at 4° C. The following morning, goatanti-human IgG conjugated with alkaline phosphatase (1:2000) was addedto the wells and incubated for two hours at 4° C. The substratepara-nitrophenyl-phosphate 104 was then added to quantify antibodybinding. The optical density (OD) values were read at 405 nm at 60 minusing a HIDEX Sense ELISA photodensity reader. Data were expressed in ODunits comparing the various subgroups.

Cell-based GnRHR-AAb Activity Assays: Activity of GnRHR AAb in IgGpurified from sera of subjects with PCOS and controls were analyzed witha GnRHR-transfected Chem-1 cell-based calcium flux assay (EurofinsScientific, USA). Briefly, Chem-1 cells were dispensed into 96-wellplates and incubated 24 h, after which assay plates were washedsufficiently with Hank's Balanced Salt Solution (HBSS) supplemented with20 mM HEPES, 2.5 mM Probenecid at pH 7.4 to remove all trace of MediaComponent. Serum-derived IgG (0.1 mg/mL) was added in the presence andabsence of the non-selective GnRHR blockade. Samples were tested intriplicate. FLUO-8®, AM Ca²⁺ dye (Cat No: 21080, AAT Bioquest, Inc.,Sunnyvale, Calif.) was prepared by dissolving 1 mg of Fluo-8 NW in 200μL of DMSO and applied to the assay plate (Ca²⁺ dye at 10 μL/10 mL issufficient for loading one plate). Excitation wave length was set at470-495 nm and emission wave length at 515-565 nm for Ca²⁺ dyes. Maximalfluorescence signal was obtained by HIDEX Sense reader.

AMH levels by Gen II ELISA: The Anti-Müllerian hormone (AMH) Gen IIELISA is an enzymatically amplified two-site immunoassay (BECKMANCOULTER, USA) with units of ng/ml. In the assay, calibrators, controls,and samples were incubated in microtitration wells which have beencoated with anti-AMH antibody. After incubation and washing, anti-AMHdetection antibody labeled with biotin was added to each well. After asecond incubation and washing step, streptavidin-horseradish peroxidase(HRP) was added to the wells. After a third incubation and washing step,the substrate tetramethylbenzidine (TMB) was added to the wells. Lastly,an acidic stopping solution was added. The degree of enzymatic turnoverof the substrate was determined by dual wavelength absorbancemeasurement at 450 nm and between 600 nm and 630 nm. The absorbancemeasured was directly proportional to the concentration of AMH in thesamples. A set of AMH calibrators was used to plot a calibration curveof absorbance versus AMH concentration. The AMH concentrations in thesamples can then be calculated from this calibration curve.

Statistical Analysis: Group data are presented as mean±SD or percent.Normally distributed groups were compared using Student t or Pearsonchi-squared tests. A ROC curve was used to assess OD as a diagnostictest for PCOS. Significance was assigned to two way values of p<0.05.

Methods Summary: Infertile PCOS subjects based on Rotterdam criteria andinfertile ovulatory controls seen at an academic fertility clinic2012-2016 were included in the study if they had stored serum prior tobeginning treatment. Serum was screened by ELISA for AAbs to GnRHR usinga synthetic 28-mer peptide from the ECL2 and ECL 1 of human GnRHR ascoating antigen. Optical density (OD) values were read at 405 nm at 60minutes. Activity of GnRHR AAb in sera and in IgG purified from sera of4 subjects with PCOS and 4 controls was analyzed with aGnRHR-transfected Chem-1 cell-based calcium flux assay. AAb-specificeffect was tested by GnRHR blockade. Group data are presented as mean±SDor percent. Groups were compared using Student t or Pearson chi-squaredtests. An ROC curve was used to assess OD as a diagnostic test for PCOS.

Results

There were no significant differences in the age of the PCOS subjectsand those with non-PCOS related infertility. There was a significantincrease in the BMI of the PCOS compared to their control subjects.

ELISA data: Each PCOS subject's serum initially was assayed by ELISA forantibodies directed toward the hECL-2 and then for ECL1 as describedabove. These assays were performed in triplicate in 384 well plates toeliminate intra-well variance. The plate included PCOS subjects andtheir closely age matched infertile but ovulatory control subjects. FIG.1A demonstrates the ELISA data for the PCOS subjects and controls thatwere age and BMI matched. There was a significant increase in theGnRHR-ECL2 ELISA OD for the PCOS versus the controls (p<0.003) (leftpanel). This assay tested sera at a 1/50 dilution, and the resulting ODwas read at 60 minutes. A repeat assay was then performed several monthslater using the same sera stored at 4° C. (right panel). This assay useda 1/25 serum dilution and was read at 30 minutes. These data werevirtually identical, with a difference in OD between the PCOS andcontrol groups of p=0.005. These data express the direct orthostericimpact of the sera on the receptor. Although the group's values weresignificantly different, there was some overlap of a number of PCOSsamples with the control group. Anti-Millerian hormone levels wereindependently assayed in a subset of these subjects (data not shown). Ifthere was a presence of either or both, there was a higher predictivevalue for a Dx of PCOS. The ELISA assay for GnRHR-ECL1-AAb for PCOSsubjects versus controls is shown in FIG. 1B. There was no evidence forAAb directed toward the ECL1. The ROC curve (FIG. 1C) for the ECL2 dataindicate a 72% sensitivity and a 55% specificity. The PCOS values byELISA were compared to the subgroups of the control ovulatory butinfertile subjects, and the PCOS ELISA values were found to be higher(p<0.01) than each of the subgroups of Tubal Factor, Male FactorInfertility (MFI), and Unexplained Infertility (FIGS. 2A and 2B). Thecombination of the PCOS and anti-Müllerian hormone in the form of a PCOSassay panel provides a superior level of discrimination in theevaluation of infertility and provides a reliable diagnostic assay forPCOS.

Activity Assays: GnRHR-transfected cells were used to examine theactivity of sera from subjects demonstrating a significant presence ofGnRHR-ECL2 Ab on ELISA. FIGS. 3A and 3B demonstrate this assay issensitive to a dosage of 10⁻⁹ M and above using the normal agonist hLHRH(Fisher, Inc) and at 10⁻⁸ M and higher using the selective agonistleuprolide (LEUPRON®, AbbVie Inc., North Chicago, Ill.).

FIGS. 4A and 4B show a composite dosage response curve (15, 75, 150, and300 mcg/ml of IgG) for IgG purified from serum from three PCOS subjectswith elevated GnRHR-ECL2 ELISA values. There was a significant increasein [Ca²⁺]_(i) observed at 75, at 150, and most dramatically at 300mcg/ml in GnRHR-transfected cells.

FIG. 5 demonstrates the effect of the GnRHR activity in the transfectedcells following exposure to sera from 3 PCOS subjects and 3 controlsubjects before and after 2-hour exposure to the specific GnRHRantagonist cetrorelix. There was no significant decrease in activity inthe control subjects, while the elevated activity in the PCOS subjectsdropped to the values observed in the control patient samples.

FIG. 6 demonstrates the effect of the agonist leuprolide on theGnRHR-transfected cell activity with buffer alone and when PCOS sera wasadded with the agonist. The effect of the sera alone was greater thanbuffer alone. Sera plus leuprolide was significantly higher than theleuprolide plus buffer. These effects were at least additive.

Results Summary: The mean GnRHR ECL2-ELISA OD subjects with documentedPCOS were significantly higher than for the control subjects. The seraand IgG purified from sera were active in the GnRHR transfected cellsand produced activity increments similar to that observed when the GnRHligand and the synthetic ligand leuprolide were used instead. Areduction in AAb activity compared to when the GnRHR selective blockercetrorelix was applied was demonstrated. There was no significant effectof the cetrorelix in the majority of control subjects.

Conclusions

An ELISA assay for GnRHR AAbs based on a target peptide derived fromECL2 hGnRHR is disclosed. The assay can be used as a diagnostic test forPCOS by detecting GnRHR AAbs. The results show that such autoantibodiesare present in a highly significant number of female subjects withclinically documented PCOS compared to the control group.

A subgroup of these subjects was examined by assessing the receptoractivation potential of the autoantibody residing in their sera and/orIgG purified from their sera using GnRHR transfected cells. Thispreparation had a reporter construct that estimates the acute rise in[Ca²⁺]_(i) and is quite specific for activation of the overexpressedGnRHR and not to any low-level expression of other GPCRs. A dosageresponse curve for GnRHR activation in the serum and for purified IgGfrom the serum was demonstrated. This response was similar to that alsoobserved using the GnRHR specific natural ligand gonadotrophin releasinghormone (GnRH) that normally activates the receptor and also to theresponse to the synthetic receptor-specific analog leuprolide, which isa potent acute agonist for this receptor. In addition, the GnRHRactivity in sera from subjects with elevated GnRHR-AAb activity wasblocked using the GnRHR antagonist cetrorelix. This specific receptorblocker allowed for the characterization of the degree of activityobserved in the sera measuring the endogenous activity minus theactivity following receptor blockade. It was observed that the elevatedactivity observed in the PCOS subjects is significantly lowered inresponse to the receptor blockade. Conversely, the majority of controlsubject activities were not suppressed from their baseline values bythis selective antagonist. This supports the concept that there waslittle if any specific autoimmune activation of the receptor in thecontrol subjects. There were a few elevated values in the “control”subjects that demonstrated some specific inhibitor-sensitivesuppression.

As shown above, evidence is provided herein that AAbs directed towardthe ECL2 of the GnRHR are significantly elevated in PCOS compared tocontrol infertile but ovulatory women. These autoantibodies willactivate the GnRHR and are therefore capable of interacting in vivo bothat the hypothalamic and anterior pituitary level. Therefore, they arecapable of disrupting the highly regulated and required pulsatile timingrequired for normal ovulatory function and play a pathogenic role inPCOS. No other Ab directed toward the GnRHR-ECL nor any Ab capable ofactivating the receptor has previously been reported.

Example 2: GnRHR AAb Binding Peptides

A Mimotope PIN ELISA assay was performed on 11 PCOS subjects and 5control subjects using sequential GnRHR-ECL2 synthetic peptides, each 8amino acid residues in length, and each one different from the precedingpeptide by addition of the next two C-terminal residues and removal ofthe last 2 N-terminal residues (see Table 1). The specific peptides werebiochemically bonded to the tip of the pin that is attached to the 96well plate cover in such a fashion that the peptides were immersed inthe buffer/sera (1:200) mixture in the well. After incubation, the pinsand wells were washed thoroughly, and the well mixture was then replacedby the anti-human-2nd AB (1:10,000) and incubated and washed again; thecolorimetric agent was then added. The wells were sequentially countedat 10 min intervals, and values were compared for analysis of O.D. foreach pin. The values and pin sequences are shown with time of reading incolor code. Results are shown in FIGS. 7-10.

TABLE 1 GnRHR-ECL2 amino acid sequence (SEQ ID NO: 1)and synthetic peptides (SEQ ID NOS: 2-12) DSSGQTKVFSQC

SFSQWWHQAFYN (SEQ ID NO: 1)                     WWHQAFYN (SEQ ID NO: 2)                  SQWWHQAF (SEQ ID NO: 3)                 SFSQWWHQ(SEQ ID NO: 4)               HCSFSQWW (SEQ ID NO: 5)             

SFSQ (SEQ ID NO: 6)           QC

SF (SEQ ID NO: 7)         FSQC

(SEQ ID NO: 8)       KVFSQCVT (SEQ ID NO: 9)     QTKVFSQC(SEQ ID NO: 10)   SGQTKVFS (SEQ ID NO: 11) DSSGQTKV (SEQ ID NO: 12)

Five PCOS subjects with two peaks with a single pin of somewhat lesseractivity between them were observed. The O.D. activity for each was >0.8in each.

Three PCOS subjects were found with a somewhat different pattern. Onehad two adjacent peaks with very high O.D. values coincident with theN-terminal first of two peaks observed in the five PCOS subjects. Theother two had relatively broad reactivity over several of the C-terminalpins with moderate to significant O.D. values. Five control subjectswere examined. Two had a single peak coincident with the C-terminalfirst peak noted in the PCOS. These single peaks were of moderate O.D.activity. Three others had either no peaks or a low activity in theregion of the second C-terminal amino acid. The whole sequence of theGnRHR-ECL2 is portrayed as SEQ ID NO:1. Overlapping Peak 1 is SEQ IDNO:8. Overlapping Peak 2 is SEQ ID NO:6.

The use of the L-peptide for each peak or, in certain non-limitingembodiments, the retro-inverso D-amino acid (RID) homolog, can be usedto identify which peak is critical for activation of the receptor. Theneach peptide separately and/or together will be preincubated with thepatient's sera for 30 minutes, and then the mixture used to test forbiological activity in the GnRHR transfected cell activity assay.CVTHCSFS (SEQ ID NO:13), comprising portions of SEQ ID NOS:6-8, is apeptide sequence that could be used to block both epitopes. The RIDhomologs can be formed in accordance with the disclosure in PatentApplication Publication No. US 2016/0222084 (published Aug. 4, 2016;U.S. Ser. No. 14/776,855, filed Sep. 15, 2015), which explains how suchretro-inverso D-amino acid homologs can be produced, and which is herebyexpressly incorporated herein by reference in its entirety.

Thus, in accordance with the present disclosure, there have beenprovided compositions, as well as methods of producing and using same,which fully satisfy the objectives and advantages set forth hereinabove.Although the present disclosure has been described in conjunction withthe specific drawings, experimentation, results, and language set forthhereinabove, it is evident that many alternatives, modifications, andvariations will be apparent to those skilled in the art. Accordingly, itis intended to embrace all such alternatives, modifications, andvariations that fall within the spirit and broad scope of the presentdisclosure.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference. In addition, thefollowing is not intended to be an Information Disclosure Statement;rather, an Information Disclosure Statement in accordance with theprovisions of 37 CFR § 1.97 will be submitted separately.

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What is claimed is:
 1. A method of therapeutically treating or reducingthe onset or occurrence of a disease or condition associated with theactivation of gonadotropin releasing hormone receptor autoantibodies(GnRHR-AAb), wherein the disease or condition is selected from the groupconsisting of Polycystic Ovary Syndrome (PCOS), a condition related toPCOS in male subjects, and a refractory cancer selected from the groupconsisting of ovarian, breast, endometrial, uterine, prostate, andtesticular cancer, the method comprising the step of: administering to asubject in need thereof a peptide selected from the group consisting ofat least one of: (1) a peptide comprising 4 to 45 amino acids, whereinthe 4 to 45 amino acids comprise the retro-inverso D-amino acid sequenceof SEQ ID NO:15; and (2) a peptide comprising 6 to 45 amino acids,wherein the 6 to 45 amino acids comprise the amino acid sequence of SEQID NO:14, at least one D-amino acid upstream of the N-terminal end ofSEQ ID NO:14, and at least one D-amino acid downstream of the C-terminalend of SEQ ID NO:14.
 2. The method of claim 1, wherein the at least oneupstream D-amino acid is immediately upstream of the N-terminal end ofSEQ ID NO:14, and the at least one downstream D-amino acid isimmediately downstream of the C-terminal end of SEQ ID NO:14.
 3. Themethod of claim 1, wherein the peptide is administered in a compositioncomprising a pharmaceutically-acceptable carrier, vehicle, or diluent.4. The method of claim 1, wherein the subject is male, and wherein thepeptide is administered in an amount effective to treat at least onecondition associated with PCOS, wherein the condition is selected fromthe group consisting of obesity, insulin resistance as manifested byType 2 diabetes mellitus, hypertension, and hyper-androgenism.
 5. Themethod of claim 1, wherein the disease or condition is a refractorycancer selected from the group consisting of ovarian, breast,endometrial, uterine, prostate, and testicular cancer.
 6. A method oftherapeutically treating or reducing the onset or occurrence ofPolycystic Ovary Syndrome (PCOS), comprising the step of: administeringto a subject in need thereof a peptide selected from the groupconsisting of at least one of: (1) a peptide comprising 4 to 45 aminoacids, wherein the 4 to 45 amino acids comprise the retro-inversoD-amino acid sequence of SEQ ID NO:15; and (2) a peptide comprising 6 to45 amino acids, wherein the 6 to 45 amino acids comprise the amino acidsequence of SEQ ID NO:14, at least one D-amino acid upstream of theN-terminal end of SEQ ID NO:14, and at least one D-amino acid downstreamof the C-terminal end of SEQ ID NO:14.
 7. The method of claim 6, whereinthe at least one upstream D-amino acid is immediately upstream of theN-terminal end of SEQ ID NO:14, and the at least one downstream D-aminoacid is immediately downstream of the C-terminal end of SEQ ID NO:14. 8.The method of claim 6, wherein the peptide is administered in acomposition comprising a pharmaceutically-acceptable carrier, vehicle,or diluent.